Peptide extract of lupine and pharmaceutical or cosmetic or nutritional composition comprising the same

ABSTRACT

The invention concerns a peptide extract of lupine (lupinus) characterised in that it has a metalloprotease inhibiting activity, in particular collagenase and gelatinase. The invention also concerns a pharmaceutical, cosmetic or nutritional composition comprising a peptide extract, optionally an inert carrier, in particular for treating humans or mammals suffering from a condition or disease related to excessive degeneration of support by a metalloprotease.

[0001] The present invention relates to a novel peptide extract whichhas antimetalloprotease activity, in particular anticollagenase andantigelatinase activity. It also relates to the pharmaceutical, cosmeticor nutraceutical compositions comprising such an extract, in particularto a pharmaceutical composition intended to treat inflammatory diseases,such as arthrosis, parodontosis or ulcers, or to the cosmeticcompositions intended to combat aging, which may or may not be actinicaging, or aging accelerated by outside attacks (tobacco, pollution,etc.).

[0002] The pharmaceutical, cosmetic or nutraceutical composition is alsointended to treat neoangiogenesis (vessel proliferation) which ispathological or unsightly (psoriasis, tumors, erythosis, acneerythematosa, rosacea, local treatment with irritants such as retanoicacid), cicatrization deficiencies, burns or the attack of dental enamel(Ch. M. Lapiere, Cours de biologie de la peau [Skin biologycourse]—COBIP INSERM U 346, Lyon 1999).

[0003] Metalloproteases are a family of zinc- and calcium-dependentendopeptidases which have the combined property of degrading the diversecomponents of connective tissue matrices (thesis by S.Charvat—Métalloprotéinases et épiderme [Metalloproteinases andepidermis], pages 101-113 No. 248-98, 1998, Lyon I).

[0004] They are classified according to the nature of their substrate:collagenase (fibrillar collagen: ex. MMP-1, -13, -8); gelatinase(denaturated collagen, gelatin: ex. MMP-2, MMP-9); stromelysins(fibronectin, proteoglycin: ex. MMP-3, MMP-10). They are used in thephysiological remodeling (low expression) or pathological remodeling ofthe extracellular matrix (strong induction).

[0005] Metalloproteases are in particular involved in the cicatrizationprocess, eliminating the damaged tissues.

[0006] MMPs may act anarchically and cause significant lesions if theiractivity is not controlled.

[0007] Moreover, it is known that metalloproteases are involved incertain biological disorders, such as inflammatory diseases, inparticular arthrosis and parodontosis (H. BIRKEDAL-HANSEN et al.,Critical Reviews in Oral Biology and Medicine, 4(2): 197: 250 (1993)),in the processes of aging, in particular linked to the action of solarradiation (MARTIN RIEGER; Allured's Cosmetics & Toiletries®, Vol. 114,No. 1/January 1999 or G. J. FISHER et al., The New England Journal ofMedicine, Vol. 337, No. 20 pp. 1419-1428, “Pathophysiology of prematureskin aging induced by ultraviolet light” and G. J. FISHER et al., theSociety for Investigative Dermatology, Inc. 1998, pp. 61-68 “Molecularmechanisms of photoaging and its prevention by retinoic acid:ultraviolet irradiation induces MAP kinase signal transduction cascadesthat induce A-1-regulated matrix metalloproteinases that degrade humanskin in vivo”) or in acute and chronic inflammations (XIE et al.; J.Biol. Chem. 273: pp. 11576-11582; 1998) and blistering diseases (toxicepidermal necrolysis), pathologies with cellular hyperproliferationduring inflammation or irritation, bedsores, burns and ulcers.

[0008] The same is true for the proliferation of neoangiogenesisendothelial cells which, in their proliferative phase duringinflammatory or pathological processes (psoriasis, tumors) need MPPs todestroy the connective tissue, in order to migrate toward other regionsand to form microtubules and capillaries (Controlling the vasculature:angiogenesis, antiangiogenesis and vascular targeting of gene therapy—T.P. D. FAN, R. JAGGAR and R. BICKNELL, TiPS—February 1995, Vol. 16;Natural Products as angiogenesis inhibitors, D. H. PAPER, Planta Medical64 (1998) pp. 686-695; Membrane-type matrix metalloproteinases in humandermal microvascular endothelial cells: expression and morphogeneticcorrelation—V. T. CHAN et al., J.I.D. 111, pp. 1153-1159, 1998; Matrixmetalloproteinases in blood vessel development in human fetal skin andin cutaneous tumors—T. V. KARELINA et al. J.I.D.; 105, 411-417, 1995;Vascular profiferation and angiogenic factors in psoriasis, J. D.CREAMER and J. N. W. N. BARKER, Clinical and Experimental Dermatology,1995, 20, pp. 6-9).

[0009] The role of inhibitors of metalloproteases, in particular ofcollagenases, gelatinases and of stromelysins, in certain treatments forthe abovementioned diseases is also known.

[0010] The object of the present invention is to provide a novelbroad-spectrum inhibitor of metalloproteases of the collagenase orgelatinase type, which allows the treatment of humans or of mammalssuffering from a condition or a disease linked to excess or pathologicaldegradation of collagen or of another extracellular supportmacroprotein, or any other diseases linked to excessive expression ofthese proteolytic enzymes.

[0011] A subject of the invention is a peptide extract of lupin(lupinus), characterized in that it has activity for inhibitingmetalloproteases, in particular collagenases or gelatinases. As avariety of lupin, mention is made in particular of the sweet white lupingenus (lupinus albus), such as the Ares variety which has a low alkaloidcontent.

[0012] A subject of the invention is in particular a novel peptideextract of lupin (lupinus), characterized in that it has an activity forinhibiting purified Clostridium histolycum collagenase on DQ-gelatin,which is in particular greater than 50% at 24 hours, for a concentrationof equal to or greater than 0.1% (w/v).

[0013] According to one variant, this peptide extract of lupin islipid-depleted.

[0014] It advantageously comprises at least 50%, preferably at least70%, preferably at least 80% of peptide.

[0015] These peptides are obtained by hydrolysis of the protein fractionof lupin.

[0016] The hydrolysis may be carried out by any suitable means, inparticular enzymatic hydrolysis.

[0017] A process for preparing such a peptide extract of lupin comprisesthe following steps:

[0018] preparing a lipid-free, ground lupin meal or a micronized lupinflour (containing lipid),

[0019] extracting the soluble protein and saccharide fractions orprecipitating with acid pH (4 or 5) depending on the isoelectric point,

[0020] optionally separating the protein fraction,

[0021] hydrolyzing the protein fraction and recovering, optionally afterfiltration, the protein extract.

[0022] The invention also relates to the protein extract which can beobtained using the abovementioned process.

[0023] In general, the invention comprises the lipid-containing lupinflours and the peptide extracts also comprising the sugars.

[0024] Preferably, the protein extract has the following amino acidcomposition (percentage by weight relative to the total weight of aminoacids). Amino acids %/total AAs ASP 11.3 GLU 23.2 SER 5.1 HIS 1.7 GLY3.4 THR 3.2 ALA 2.8 ARG 10.3 TYR 6.1 CYS-CYS 2.4 VAL 3.8 MET 0.2 PHE 7.0ILE 3.3 LEU 7.9 LYS 3.7 PRO 4.4

[0025] A subject of the invention is also a pharmaceutical, cosmetic ornutraceutical composition comprising a peptide extract as describedabove and, optionally, a suitable physiologically acceptable inertvehicle.

[0026] Such a pharmaceutical or dermocosmetic and nutraceuticalcomposition is intended in particular for treating humans or mammalssuffering from a condition or disease linked to excessive destruction ofcollagen and/or excessive destruction of support tissues. Such acomposition may be used by way of prevention or cure.

[0027] Among these conditions or diseases, mention is made, for example,of arthrosis, periodontal diseases, diseases linked to skin lesions,inflammatory diseases, tumor-related or pathological neoangiogenesis(erythrosis, acne erythematosa, telangiectasia, rosacea, psoriasis,etc.), cicatrization deficiency, ulcers, burns, blistering diseases andthe attack of dental enamel.

[0028] The invention also relates to cosmetic compositions for treatingskin lesions due to aging, such as lesions caused by the action of solarradiation (photoaging), the intrinsic deleterious effects of the skin orthe deleterious effects of tobacco.

[0029] The pharmaceutical, dermocosmetic or cosmetic compositions are,according to one variant, in the form of a formulation for topicalapplication. A subject of the invention is therefore a method forcosmetic treatment, comprising applying such a composition on thesurface of the skin of an individual.

[0030] The peptide extract according to the invention may also beincorporated into or formulated in a polymeric vehicle or a deliverysystem, for topical or local use, such as in the case of treating aperiodontal disease, so as to be delivered directly into the periodontalpocket.

[0031] According to another variant, the pharmaceutical compositions arein the form of a formulation for oral administration.

[0032] These compositions may, in general, be formulated in the form oftablets, of capsules or of ointment.

[0033] In the case of nutraceutical compositions or food compositions,the forms conventionally used may be employed.

[0034] The invention is now illustrated using the examples ofimplementation described hereinafter, by way of illustration.

[0035] I. Preparation of Peptide Extracts of Lupin

[0036] I.1.—Extract A

[0037] Extraction and Purification of Lupin Proteins

[0038] This step comprises aqueous solubilization of the fractionsoluble at alkaline pH, followed by separation from the insolublecomponents:

[0039] Using the lipid-free ground lupin meal, the proteins areextracted at pH 9.0 (pH adjusted by adding sodium hydroxide) with aflour/water ratio equal to {fraction (1/10)} (w/w). The solution isincubated with stirring at room temperature for one hour. The insolubleportion of the meal is then separated from the soluble portion byspin-drying. The cake obtained is washed. The soluble fractioncontaining the soluble sugars and proteins is diafiltered on anultrafiltration module with a cutoff threshold of 10 000 Daltons, inorder to separate the proteins (retentate) from the soluble sugars(ultrafiltrate).

[0040] Production and Purification of Peptides by Enzymatic Hydrolysis:

[0041] The ultrafiltration retentate containing the proteins is adjustedto the concentration of 100 g/l and then hydrolyzed at pH 8.0 in thepresence of Alcalase® (NOVO NORDISK) at 55° C. for approximately 3 h.After hydrolysis, the enzyme is denatured by hydrolysis for 15 min at85° C. As soon as the solution has cooled down, it is neutralized byadding hydrochloric acid. The peptides obtained are purified bydiafiltration on an ultrafiltration module with a cutoff threshold of 10000 Daltons. The solution obtained is then nanofiltered in order todesalify (eliminate the sodium chloride) and concentrate the peptidefraction. The peptide solution is finally decolorized using 3% activecharcoal (1 hour at 50° C.), the charcoal being removed by filtration.

[0042] Sterilization and Packaging of the Peptide Fraction:

[0043] Before packaging, the solution is microfiltered (0.2 μm)sterilely and then distributed into sterile containers at theconcentration of 10%, in the presence of preserving agents.

[0044] I.2—Extract B

[0045] The peptide extract B is obtained according to the describedmethod implemented for obtaining extract A, except that thedecolorization step is deleted.

[0046] I.3—Extract C

[0047] The peptide extract of lupin C is obtained according to thedescribed method implemented for obtaining extract A, except that thepurification, ultrafiltration and decolorizing steps are deleted.

[0048] II—Analysis of Peptide Extract A

[0049] The dry extract is then analyzed.

[0050] Presentation: Appearance: nonhygroscopic homogeneous powderColor: off-white Amount: 5 g Chemical composition: Total of sugarcontent (test  <1% using anthrone): Chloride content (SIGMA kit    6%ref: 955-30): Water content (100° C., 4 h):    8% maximum Peptidecontent:   85% Characterization pH (solution at 20 g/l): 7.06 Solubility(osmozed water): >100 g/l

[0051] TABLE 1 Amino acid composition of the hydrolysate Amino M.W.Conc. in Conc. in % in %/total acids A.A. mM mg/l powder AAs ASP 133.12.078 276.582 9.9 11.3 GLU 147.1 3.858 567.438 20.3 23.2 SER 105.1 1.196125.647 4.5 5.1 HIS 155.2 0.270 41.904 1.5 1.7 GLY 75.1 1.114 83.624 3.03.4 THR 119.1 0.664 79.023 2.8 3.2 ALA 89.1 0.763 67.983 2.4 2.8 ARG174.2 1.447 251.980 9.0 10.3 TYR 181.2 0.829 150.215 5.4 6.1 CYS-CYS240.3 0.247 59.234 2.1 2.4 VAL 117.1 0.792 92.743 3.3 3.8 MET 149.20.029 4.327 0.2 0.2 PHE 165.2 1.044 172.469 6.2 7.0 ILE 131.2 0.62181.410 2.9 3.3 LEU 131.2 1.481 194.307 6.9 7.9 LYS 146.2 0.626 91.4483.3 3.7 PRO 115.1 0.935 107.619 3.8 4.4 Total 2447.952 87.4%

[0052] III—Anticollagenase and Antigelatinolytic Activity of the LupinPeptides—Extract A, In Vitro

[0053] The anticollagenase activity was measured in vitro in abiochemical model of the screening type, based on the use of a purifiedcollagenase and of the substrate for the latter, gelatin conjugated tofluorescein (EnzChek™ gelatinase/collagenase kit, MOLECULAR PROBES). Thecollagenase purified from Clostridium histolyticum was supplied in theEnzChek™ gelatinase/collagenase kit (MOLECULAR PROBES). This enzymes hasa double functionality on collagen IV (epidermis/dermis basal membrane)and gelatin.

[0054] The DQ-gelatin purified from porcine skin and conjugated tofluorescein was supplied in the EnzChek™ gelatinase/collagenase kit(MOLECULAR PROBES).

[0055] The reaction buffer, consisting of 0.05 M of Tris-HCl, 0.15 MNaCl, 5 mM of CaCl₂, and 0.2 mM of sodium azide (pH 7.6) was supplied inthe EnzChek™ gelatinase/collagenase kit (MOLECULAR PROBES).

[0056] The peptide extract was solubilized in the reaction buffer. Itwas tested at 0.004; 0.02; 0.04; 0.2 and 0.4% (w/v).

[0057] The dilutions of the test extracts were incubated with DQ-gelatinat 1 mg/ml and collagenase at 0.2 Ru/ml for 1 hour, 2 hours and 24 hoursat room temperature.

[0058] A control, corresponding to the “collagenase+DQ-gelatin” mixture,was incubated in parallel.

[0059] For each experimental condition, samples, subsequently named“samples without enzyme”, were incubated in the presence of DQ-gelatinand in the absence of collagenase.

[0060] Each experimental condition was performed in triplicate.

[0061] After 1 hour, 2 hours and 24 hours, the signal corresponding tothe degradation of the DQ-gelatin was measured by fluorimetry(excitation: 485 nm and emission: 595 nm). For each sample, the valueobtained for the “samples without enzyme” was attracted.

[0062] The results were expressed as fluorescence units per sample andas percentage variation relative to the control group.

[0063] The groups of data (control group and treated groups) werecompared by factor analysis of variance (ANOVA 1, p<0.05), followed by aDunnett's test. The effect of the extracts was thus compared to thatobtained in the control group.

[0064] The peptide extract tested from 0.004 to 0.2% (w/v) had adose-dependent anticollagenase and antigelatinolytic activity. Theeffect was maximal at the 24 hour time point, as indicated in Table 2below. TABLE 2 Control 0.004 0.02 0.04 0.2 0.4 Incubation time = 1 hour16237 14161 11890 11205 11249 9434 14329 13561 11161 10863 9840 754415636 13965 11757 11344 11387 8878 15401  13896  11603*  11137*  10825* 8619* +/− +/− +/− +/− +/− +/− 976 306 388 248 856 971 100% 89 75 73 7357 Incubation time = 2 hours 24776 20526 13689 11000 7617 6853 2251619597 6710 10406 6072 4933 23779 20144 13148 11349 7824 6467 23690 20089*  11182*  10918*  7171*  6084* +/− +/− +/− +/− +/− +/− 1133 4673883 477 957 1016 100% 85 55 48 33 27 Incubation time = 24 hours 3165312655 2583 2378 524 1154 29536 11531 1487 1442 484 467 29745 13008 26572713 693 927 30311  12398*  2242*  2178*  567*  849* +/− +/− +/− +/− +/−+/− 1167 771 655 659 111 350 100% 41 7 7 2 3

[0065] In conclusion, under the experimental conditions selected, theprotein extract tested between 0.004 and 0.4% (w/v) had a dose-dependentantigelatinase/collagenase activity. An excellent effect/dose/time ratiofor the lupin peptides compared to the aspecific collagenase is inparticular noted: at 24 hours, for example, 0.04% inhibits 93% of thegelatinolytic activity of the clostridium collagenase, at 2 hours: 52%.

[0066] Other tests with the same peptide extract were carried out atconcentrations of 0.01; 0.05; 0.1; 0.5 and 1% (w/v) and the results aregiven in table 3 below and also in the attached FIG. 1. This figurerepresents the kinetics of inhibition of the aspecificcollagenase—influence of the concentration of peptide extract. They-axis represents gelatinolytic activity (%), the x-axis representsincubation time (h). TABLE 3 Concentration of extract A (% w/v)/Incubation gelatinolytic activity as (%) time (h) 0.01 0.05 0.1 0.5 1 189 75 73 73 57 2 85 55 48 33 27 4 79 38 29 14 12 6 69 27 21 8 9 24 41 77 2 3

[0067] The aspecific inhibitor 1,10-phenanthroline was used in all thetests as a reference anti-MMP product. The results obtained were inaccordance with those expected and validated the tests.

[0068] IV—Specific Anticollagenase Activity of the LupinPeptides—Extract A, on a Human Organotypic Model

[0069] Skin aging is characterized, inter alia, by a modification of thecutaneous mechanical properties of the skin, subsequent to degradationof the collagen fibers of the dermis and of other macroproteins. Thisdegradation involves endogenous collagenases (Grymes, R. A., KronbergerA. and Bauer E. A.—Collagenases in disease—in “Connective TissueDiseases of the skin” Eds. Lapière C. M. and Krieg T., 1993, 69-85; G.Fischer and J. Voorhees “Pathiophysiology of premature skin aginginduced by ultraviolet light” and “Molecular mechanisms of photoagingand its prevention by retinoic acid: ultraviolet irradiation induces MAPkinase signal transduction cascades that induce A-1-regulated matrixmetalloproteinases that degrade human skin in vivo”).

[0070] Products which have anticollagenase activity can be envisaged forcombating the signs of skin aging.

[0071] The anticollagenase activity of a test product can be studied invitro in an organotypic model of human skin. The principle of the testis as follows: application of purified collagenase to sections isaccompanied by degradation of the endogenous collagen fibers. Thecollagen fibers are then stained with Masson trichrome. The digestion ofthe endogenous collagen fibers by the purified collagenase is evaluatedqualitatively by morphological observation and quantitatively by imageanalysis. A product which has anticollagenase activity will partially ortotally preserve the integrity of the collagen fibers placed in thepresence of the enzyme.

[0072] The test products were stored at +4° C. until they were used.

[0073] Three dilutions were tested: 0.01; 0.1 and 1% (v/v).

[0074] Phosphoramidon, used as a reference product, came from SIGMA.

[0075] The purified collagenase (type III, fraction A) came from SIGMA.

[0076] The medium for incubating the human skin sections, subsequentlynamed “vehicle” was 0.15 M Tris HCl buffer, pH 7.5, containing 0.01 M ofcalcium chloride.

[0077] The reagents, of analytical quality, came from CARLO ERBA, GIBCOor SIGMA, unless otherwise stated.

[0078] The skin sections were prepared from waste from an operation,recovered after abdominal plastic surgery. The individual was a30-year-old woman. Skin explants 4 cm in diameter were prepared. Theywere placed on a cork support and frozen at −80° C. 6 μm-thicktransverse sections were prepared using a cryomicrotome. They were fixedon glass slides and kept hydrated with the vehicle during the test.

[0079] The test samples were all taken up in ethanol before beingdiluted in the test buffer.

[0080] The final concentration of ethanol was maintained constant andequal to 0.1% (v/v) in the two weakest dilutions of the peptide extract(0.01 and 0.1% v/v);

[0081] It was maintained constant and equal to 1% (v/v) in the strongestdilution (1%, v/v).

[0082] “Ethanol controls” at 0.1 and 1% (v/v) were prepared.

[0083] The phosphoramidon was taken up directly in the vehicle.

[0084]  The peptide extract was tested at 0.01; 0.1 and 1% (w/v).

[0085]  The phosphoramidon was tested at M.

[0086] The following samples were therefore present:

[0087] Extract: buffer; ethanol (0.1% v/v or 1% v/v);

[0088] extracted enzyme (0.01; 0.1 and 1%, w/v);

[0089] Enzyme control: buffer; ethanol (0.1%, v/v);

[0090] enzyme;

[0091] Ethanol control (without enzyme): buffer; ethanol (0.1% or 1%,v/v);

[0092] Ref. product: buffer; ethanol; enzyme; 10⁻³ M phosphoramidon.

[0093] The dilutions of the test products, of the ethanol and of thereference product were placed on to the skin sections, at 100 μl persection, and pre-incubated for 10 minutes at 37° C. Strips of filterpaper (0.16 cm² surface area) soaked with vehicle alone (control withoutenzyme) or containing collagenase at 50 international units (IU)/ml(enzyme control) were then placed over the sections. The slides wereplaced in a humid chamber at 37° C. for three hours.

[0094] After incubation, the sections were rinsed with the incubationmedium and stained with Masson trichrome. The activity of the enzyme inthe presence and absence of the extract, of the ethanol or of thereference product was evaluated by microscopic observation and scoredaccording to the following scheme: 0: zero enzymatic digestion +: weakenzymatic digestion ++: average enzymatic digestion +++: strongenzymatic digestion.

[0095] Photographs of the sections were taken.

[0096] The activity of the collagenase in the presence and absence ofthe test products, of the ethanol or of the reference product wasevaluated by image analysis. The image of the stained sections wasdigitalized on a video screen; the image analysis software (IMAGENIA2000, BIOCOM®) made it possible to calculate the surface area occupiedby the intact collagen fibers. The results were expressed as apercentage of intact collagen fibers per optical field.

[0097] The percentage inhibition of the collagenase activity of theextracts at various concentrations of the ethanol and of the referenceproduct was calculated using the following formulae:

[0098] For the reference product (directly taken up in the vehicle), thepercentage inhibition is calculated using the following formula:$\frac{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ref}\quad {product}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {control} \times 100}\end{matrix}\quad}{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ethanol}\quad {control}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {control}}\end{matrix}}$

[0099] For the extracts diluted in the vehicle containing 0.1% (v/v) ofethanol, the percentage inhibition is calculated using the followingformula: $\frac{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ref}\quad {product}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {control} \times 100}\end{matrix}\quad}{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ethanol}\quad {control}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {control}}\end{matrix}}$

[0100] For the extracts diluted in the vehicle containing 1% (v/v) ofethanol, the percentage inhibition is calculated using the followingformula: $\frac{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ref}\quad {product}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {control} \times 100}\end{matrix}\quad}{\begin{matrix}{{\left( {\% \quad {collagen}\quad {fibers}} \right){ethanol}\quad {control}} -} \\{\left( {\% \quad {collagen}\quad {fibers}} \right){enzyme}\quad {{control}.}}\end{matrix}}$

[0101] The anticollagenase activity of the extract at variousconcentrations was studied in an organotypic model of human skin.

[0102] In the absence of collagenase (vehicle control), the collagenfibers were intact. In the presence of collagenase (enzyme control), thecollagen fibers were almost totally degraded. This result was expectedand validated the test.

[0103] The phosphoramidon at 10⁻³ M, used as a reference product,inhibited the collagenase activity by 16%. This result was expected andcompleted the validation of the test.

[0104] The ethanol, used as an intermediate diluent of the test productswas tested at 0.1 and 1% (v/v). It had no effect on the degradation ofthe collagen fibers by the collagenase.

[0105] The peptide extract tested at 0.01; 0.1 and 1% (w/v) inhibitedthe activity of the collagenase by 2, 24 and 65%, respectively. Themorphological observation gave the same result.

[0106] In conclusion, under the experimental conditions selected,peptide extract A had considerable anticollagenase activity at lowconcentrations.

[0107] The results of inhibition, for the peptide extract, for theethanol and for the phosphoramidon, on the digestion of dermal collagenfibers by collagenase are given in the table below: TABLE 4 Enzymatic %inhibition of digestion the collagenase Experimental (morphologicalactivity (image condition Concentration observation) analysis) Vehiclecontrol — 0 − Enzyme control 50 IV/ml +++ 0 Phosphoramidon 10⁻³ + + (M)Ethanol 0.1 +++ 0 (%, v/v) 1 +++ 0 Peptide extract 0.01 +++ 2 (%, v/v)0.1 ++ 24 1 + 65

[0108] V—Antimetalloprotease MMP-2 and MMP-9 Activity of the LupinPeptides—Extracts A, B and C

[0109] MMP-2, or gelatinase A, and MMP-9, or gelatinase B, aremetalloproteases which degrade specific components of the extracellularmatrix: MMP-2 degrades gelatin (=denatured collagen), collagens I, IV,VII and XI, fibronectin, laminin and elastin; MMP-9 degrades gelatin,collagens IV, V and XIV and elastin. They play an important role inphotoaging and in the proliferation of endothelial cells.

[0110] The anti-MMP-2 and anti-MMP-9 activity of the test products wasmeasured in vitro in a biochemical model of the screening type, based onthe use of a purified human MMP-2 and of a recombinant human MMP-9 andof the substrate for the latter, gelatin conjugated to fluorescein(EnzChek™ gelatinase/collagenase kit, MOLECULAR PROBES).

[0111] The MMP-2 purified from human fibrosarcoma came from BOEHRINGERMANNHEIM.

[0112] The recombinant human MMP-9 came from R&D SYSTEMS.

[0113] The DQ-gelatin purified from porcine skin and conjugated tofluorescein was supplied in the EnzChek™ gelatinase/collagenase kit(MOLECULAR PROBES).

[0114] The reaction buffer for studying the activity of MMP-2 (RBfl)consisted of 50 mM of Tris-HCl, 0.05% (w/v) of Triton X100 and 5 mM ofCaCl₂, pH 7.5.

[0115] The reaction buffer for studying the activity of MMP-9 (RBf2)consisted of 50 mM of Tris-HCl, 0.05% (w/v) of Brij 35 and 5 mM ofCaCl₂, pH 7.4.

[0116] Preparation of the Test Products and of the Reference Product

[0117] 1,10-Phenanthroline was solubilized in the reaction buffers RBf1and RBf2. It was tested at 8 and 80 μg/ml.

[0118] Peptide extracts A, B and C were solubilized in the reactionbuffers RBf1 and RBf2. They were tested at 0.01; 0.1 and 1% (w/v).

[0119] MMP-2

[0120] Before being used, the MMP-2 was activated by incubation for 30minutes at 37° C. in the presence of APMA diluted to 2.5 mM in thebuffer RBf1.

[0121] The dilutions of the test products or of the reference productwere incubated with the DQ-gelatin, diluted to 25 μg/ml, and theactivated MMP-2, diluted to 1.25 μg/ml, for 24 hours at 37° C.

[0122] A control corresponding to the “MMP-2+DQ-gelatin” mixture, wasincubated in parallel.

[0123] For each experimental condition, samples, subsequently named“samples without enzyme”, were incubated in the presence of DQ-gelatinand in the absence of activated MMP-2. These samples made it possible tomeasure the interference of the test products with the method forevaluating the effects (fluorimetry).

[0124] Each experimental condition was performed in triplicate.

[0125] MMP-9

[0126] The dilutions of the test products or of the reference productwere incubated with the DQ-gelatin, diluted to 25 μg/ml, and the MMP-9,diluted to 0.25 μg/ml, for 24 hours at 37° C.

[0127] A control corresponding to the “MMP-9+DQ-gelatin” mixture, wasincubated in parallel.

[0128] For each experimental condition, samples, subsequently named“samples without enzyme”, were incubated in the presence of DQ-gelatinand in the absence of MMP-9. These samples made it possible to measurethe interference of the test products with the method for evaluating theeffects (fluorimetry).

[0129] Each experimental condition was performed in triplicate.

[0130] After 24 hours, the signal corresponding to the degradation ofthe DQ-gelatin was measured by fluorimetry (excitation: 485 nm andemission: 595 nm) For each sample, the value obtained for the “sampleswithout enzyme” was subtracted.

[0131] The results were expressed as fluorescence units per sample andas percentage variation relative to the control group.

[0132] The groups of data (control group and treated groups) werecompared by onefactor analysis of variance (ANOVA 1, p<0.05), followedby a Dunnett's test.

[0133] The results are given below: V.1 - Anti-MMP-2 activity MMP2activity (as %) (1)/Concentration (% v/v) of 10% by weight solution ofPeptide peptide extract extract Control 0.01 0.1 1 C 100 119 111 43 A100 152 151 68 B 100 110  98 77

[0134] Conclusion:

[0135] The 10% by weight solution of extract of lupin C, tested at 0.01and 0.1% (v/v) has no anti-MMP-2 activity. When tested at 1% (v/v), itinhibits the MMP-2 by 57%.

[0136] The 10% by weight solution of extract of lupin A, tested at 0.01to 0.1% (v/v) has no anti-MMP-2 activity. When tested at 1% (v/v), itinhibits the MMP-2 by 32%.

[0137] The 10% by weight solution of extract B, tested at 1%, inhibitsthe MMP-2 by 23%.

[0138] The phenanthroline, tested at 8 and 80 μg/ml, inhibits theactivity of the MMP-2 by 32 and 73%, respectively. This result, whichwas expected, validates the test. V.2 - Anti-MMP-9 activity MMP-9activity (as %) (1)/Concentration (% v/v) of 10% by weight solutionPeptide of peptide extract extract Control 0.01 0.1 1 A 100 143 143 61 B100 146 129 27

[0139] Conclusion:

[0140] The 10% by weight solution of extract of lupin A, tested at 0.01and 0.1% (v/v) has no anti-MMP-9 activity. When tested at 1% (v/v), itinhibits the MMP-9 by 39%.

[0141] The 10% by weight solution of extract of lupin B, tested at 0.01and 0.1% (v/v) has no anti-MMP-9 activity. When tested at 1% (v/v), itinhibits the MMP-9 by 73%.

[0142] The phenanthroline, tested at 8 and 80 μg/ml, inhibits theactivity of the MMP-9 by 80 and 76%, respectively. This result, whichwas expected, validates the test.

[0143] VI—Evaluation of the Effect of the Lupin Peptides on the Amountof MMP-1-9 and -3 in Human Fibroblasts Irradiated with UVA Rays

[0144] The study was carried out on human dermal fibroblasts inmonolayer culture. The cells were pre-incubated for 1 hour at 37° C. inthe presence of the reference products and of the test product. Then,the cells were irradiated with a single dose of 10 J/cm² of UVA, in thepresence of the product to be tested and of the reference products.

[0145] Immediately after irradiation, the cells were incubated for 48 hat 37° C., still in the presence of the product to be tested and of thereference products.

[0146] The various MMPs are tested in the culture media using specificELISA kits (Amersham).

[0147] Reference Products and Test Product

[0148] 1,10-Phenanthroline, which is a nonspecific inhibitor of MMPs,and was tested at 80 μg/ml, was used as a reference product.

[0149] A 10 μg/ml retinoic acid+10 ng/ml EGF mixture was used for itsabilities to induce TIMP1 (TIMP1, Tissue Inhibitor of MMP, physiologicalinhibitor of MMPs).

[0150] A stock solution containing 10% (w/v) of lupin peptides wasprepared in deionized water. Using this solution, dilutions wereprepared in the fibroblast culture medium. The peptide extract of lupinwas tested at 0.5; 1 and 2% (v/v).

[0151] Irradiated and nonirradiated control cultures were incubated inparallel, in the absence of the reference products and of the testproduct.

[0152] Processing of Data

[0153] The groups of data (control group and treated groups) werecompared by one-factor analysis of variance (ANOVA 1, p<0.05), followedby a Dunnett's test. The effects of the reference products and of thetest product were thus compared to that obtained in the “irradiatedcells” group.

[0154] Results:

[0155] They are given in the table below and the results are expressedas % relative to the “irradiated cells” group. Production of MMP (as %)Irradiated cells + lupin Control Irradiated peptides (%, v/v) cellscells 0.5 1 2 MMP-1 24 100 4 3 3 MMP-9 41 100 17 2 0 MMP-3 88 100 3 0 2

[0156] Conclusions:

[0157] The 1,10-phenanthroline, tested at 80 μg/ml, inhibited by 99% thesecretion of MMP-1, by 92% the secretion of MMP-9 and by 97% thesecretion of MMP-3, by the irradiated fibroblasts.

[0158] The 10 μM retinoic acid+10 ng/ml EGF mixture inhibited by 58% thesecretion of MMP-1, by 67% the secretion of MMP-9 and by 44% thesecretion of MMP-3, by the irradiated fibroblasts.

[0159] These results were expected and validated the reactivity of thetest system.

[0160] The irradiation at the dose of 10 J/cm² increased by a factor of4.10; 2.42 and 1.13, the respective amounts of MMP-1, -9 and -3 secretedby the fibroblasts. These results were expected and validated the testsystem with regard to the induction of MMP-1, -9 and -3 by UVAradiation.

[0161] The peptide extract of lupin, tested at 0.5, 1 and 2% (v/v),decreased by 96, 97 and 97%, respectively, the amount of MMP-1 secretedby the fibroblasts (p<0.05).

[0162] The peptide extract of lupin, tested at 0.5, 1 and 2%, decreasedby 83, 98 and 100%, respectively, the amount of MMP-9 secreted by thefibroblasts (p<0.05).

[0163] The peptide extract of lupin, tested at 0.5, 1 and 2%, decreasedby 97, 100 and 98%, respectively, the amount of MMP-3 secreted by thefibroblasts (p<0.05).

[0164] Thus, under the experimental conditions selected, the peptideextract of lupin has considerable inhibitory properties with respect tothe production of MMP-1, -9 and -3 by UVA-irradiated human dermalfibroblasts.

[0165] VII—Examples of Formulae for Topical Use:

[0166] The percentages are expressed as total weight of the composition.The peptide extract of lupin is used in the form of a 10% by weightaqueous solution according to the invention or of a lyophilized powdernamed powder form peptide extract. 1. Cream acting against red blotches,for normal skin Water q.s. for 100.000  Pentaerythrityl tetraoctanoate15.0 to 5.0  Glyceryl stearate 10.0 to 2.0  Isodecyl neopentanoate 10.0to 2.0  Propylene glycol 1.0 to 3.0 Dextrin 1.0 to 3.0 Cyclomethicone1.0 to 3.0 Soybean (soybean glycine) extract  0.1 to 10.0 Peptideextract of lupin  0.1 to 10.0 (10% aqueous solution) Titanium dioxide1.0 to 3.0 Candelilla wax (Euphorbia Cerifora) 1.0 to 3.0 Rice starch(Oriza Sativa) 1.0 to 3.0 Unsaponifiable soybean 0.01 to 10.0 (soybeanglycine) oil Caprylic/capric acid triglycerides 0.5 to 5.0 PEG-100stearate 0.5 to 5.0 Sophora Japonica extract  0.1 to 10.00 Stearic acid0.5 to 1.0 Tocopheryl acetate 0.1 to 1.0 Phenoxyethanol 0.1 to 1.0 CI77891 0.1 to 1.0 Xanthan gum 0.1 to 0.5 Dimethiconol 0.1 to 0.5Polyacrylamide 0.1 to 0.5 Mica 0.1 to 0.5 Ceteareth-20 0.1 to 0.5Chlorphenesine 0.1 to 0.5 Carbomer 0.1 to 0.5 Octyl palmitate 0.1 to 0.5Tromethamine 0.1 to 0.5 Beeswax 0.1 to 0.5 C13-14 Isoparaffin 0.1 to 0.5DEA cetyl phosphate 0.1 to 0.5 Cetyl alcohol 0.1 to 0.5 Glucose 0.1 to0.5 Fragrance 0.1 to 0.5 Disodium EDTA 0.1 to 0.5 2. Antiagingpreventive cream % Water q.s. for 100.00000 Pentaerythrityltetraoctanoate  3.0 to 15.0 Isodecyl neopentanoate  3.0 to 15.0 Squalane 1.0 to 10.0 Dextrin  1.0 to 10.0 Cyclomethicone  1.0 to 10.0 Cetearylalcohol  1.0 to 10.0 Peptide extract of lupin  0.1 to 10.0 (10% aqueoussolution) Ascorbyl glucoside  0.1 to 10.0 Glycerol  1.0 to 10.0Laureth-23  1.0 to 10.0 Myristyl myristate  1.0 to 10.0Cyclopentasiloxane  1.0 to 10.0 Nylon-6  1.0 to 10.0 Avocado furan 0.01to 10.0 Phenoxyethanol 0.1 to 1.0 Cetearyl glucoside 0.1 to 1.0Fragrance 0.1 to 1.0 Beeswax 0.1 to 1.0 Methylparabene 0.1 to 0.5 Sodiumcitrate 0.1 to 0.5 Dimethiconol 0.1 to 0.5 Glyceryl stearate 0.1 to 0.5Disodium EDTA 0.1 to 0.5 Propylparabene 0.1 to 0.5 Sodium hydroxide 0.1to 0.5 Acrylate/C10-30 alkyl acrylate 0.1 to 0.5 crosspolymers Xanthamgum 0.1 to 0.5 Glucose 0.1 to 0.5 3. Antiaging cream for mature skinWater q.s. for 100.0000  Pentaerythrityl tetraoctanoate  1.0 to 10.0Isodecyl neopentanoate  1.0 to 10.0 Hydrogenated cocoglycerides  1.0 to10.0 Simmondsia Chinensis (jojoba) seed  1.0 to 10.0 oil Squalane  1.0to 10.0 Glycerol  1.0 to 10.0 Cyclomethicone 1.0 to 5.0 Cetearyl alcohol1.0 to 5.0 Myristyl myristate 1.0 to 5.0 Laureth-23 1.0 to 5.0 Silica1.0 to 5.0 Peptide extract of lupin  0.1 to 10.0 (10% aqueous solution)Sclerotium gum 0.1 to 1.0 Avocado furan 0.01 to 10.0 Salicylic acid  0.1to 10.0 Beeswax  0.1 to 10.0 Polyacrylamide 0.1 to 1.0 Phenoxyethanol0.1 to 1.0 Glyceryl stearate 0.1 to 1.0 Retinol palmitate 0.01 to 5.0 Cetearyl glucoside 0.01 to 5.0  Nylon-6 0.01 to 5.0  Titanium dioxide0.01 to 5.0  Fragrance 0.1 to 5.0 Tocopheryl acetate 0.1 to 5.0Potassium sorbate 0.1 to 5.0 Methylparabene 0.1 to 5.0 C13-14Isoparaffin 0.1 to 5.0 CI 77891 0.1 to 5.0 Dimethiconol 0.1 to 5.0Propylparabene 0.1 to 5.0 Sodium hydroxide 0.1 to 5.0 Laureth-7 0.1 to5.0 Cetearyl alcohol 0.1 to 5.0 Cetyl palmitate 0.1 to 5.0Cocoglycerides 0.1 to 5.0 Disodiuin EDTA 0.1 to 0.5 CI 77491 0.1 to 0.5Citric acid 0.1 to 0.5 4. Cream acting against red blotches, for dry tovery dry skin Water q.s. for 100.00000 Petrolatum  1.0 to 10.0Hydrogenated cocoglycerides  1.0 to 10.0 Isodecyl neopentanoate  1.0 to10.0 Simmondsia Chinensis (jojoba) 1.0 to 2.0 oil Butylene glycol 1.0 to5.0 Cetearyl alcohol 1.0 to 5.0 Glycerol  1.0 to 10.0 Squalane  1.0 to10.0 Peptide extract of lupin  0.1 to 10.0 (10% aqueous solution)Laureth-23  1.0 to 10.0 Titanium dioxide  1.0 to 10.0 Unsaponifiablesoybean glycine  0.1 to 10.0 (soybean) oil Caprylic/capric acidtriglycerides 1.0 to 5.0 Phenoxyethanol 0.1 to 1.0 Cetearyl glucoside0.1 to 1.0 Soybean seed extract  0.1 to 10.0 Fragrance 0.1 to 1.0Sophora Japonica  0.1 to 10.0 Tocopheryl acetate 0.1 to 1.0 Candelillawax 0.1 to 1.0 CI 77891 0.1 to 0.5 Methylparabene 0.1 to 0.5 Mica 0.1 to0.5 Propylparabene 0.1 to 0.5 Ethylhexyl palmitate 0.1 to 0.5Chlorphenesine 0.1 to 0.3 Acrylate/C10-30 alkyl acrylate 0.1 to 0.3crosspolymers Xantham gum 0.1 to 0.3 Disodium EDTA 0.1 to 0.3 Sodiumhydroxide 0.1 to 0.3

[0167] VIII—Examples of Mouthwash Solutions

[0168] The percentages are expressed as total weight of the composition.The peptide extract of lupin is used in the form of a 10% by weightaqueous solution according to the invention or of a lyophilized powdernamed powder form peptide extract. 1. 10% aqueous solution peptideextract of lupin + antiseptic (Triclosan) + antiplaque agent (GantrezS97BF ®) Peptide extract of lupin 2 Ethyl alcohol 10 Glycerol 10Hydrogenated castor oil 0.5. ethoxylated at 40 mol EO (Cremophor co410)Poly(methyl vinyl ether/maleic 0.2 acid) (Gantrez S97BF ®) Sodiumhydroxide 0.15 Sodium fluoride 0.05 Cinnamon-mint flavoring 0.1Triclosan 0.03 Zinc chloride 0.01 Sodium saccharin 0.01 Coloring C.I.16255 (E 124) 0.0025 Purified water q.s. for 100 2. 10% aqueous solutionpeptide extract of lupin + antiseptic Peptide extract of lupin 2 Ethylalcohol 10 Glycerol 10 Hydrogenated castor oil 0.3 ethoxylated at 40 molEO (Cremophor co410) Sodium fluoride 0.05 Cinnamon-mint flavoring 0.1Triclosan 0.03 Zinc chloride 0.01 Sodium saccharin 0.01 Coloring C.I.16255 (E 124) 0.0025 Purified water q.s. for 100 3. Powder form peptideextract of lupin + antiseptic (cetylpyridinium chloride) Peptide extractof lupin 1 Ethyl alcohol 10 Glycerol 8 Hydrogenated castor oil 0.1ethoxylated at 40 mol EO (Cremophor co410) Sodium fluoride 0.05Cinnamon-mint flavoring 0.1 Cetylpyridinium chloride 0.05 Zinc chloride0.01 Sodium saccharin 0.01 Coloring C.I. 16255 (E 124) 0.0025 Purifiedwater q.s. for 100

[0169] IX—Examples of Toothpastes

[0170] The percentages are expressed as total weight of the composition.The peptide extract of lupin is used in the form of a 10% by weightaqueous solution according to the invention or of a lyophilized powdernamed powder form peptide extract. 1. Powder form peptide extract oflupin + fluorides Peptide extract of lupin 1 Sodium monofluorophosphate0.75 Sodium fluoride 0.10 70% sorbitol 35 Synthetic silica with strong13 abrasive power Synthetic silica with weak 5 abrasive power Sodiumcarboxymethylcellulose 1.6 Sodium lauryl sulfate 1 Mentholated flavoring0.85 Titanium oxide 0.5 Sodium hydroxide lye 0.5 Sodium cyclamate 0.3Menthol 0.15 Sodium saccharin 0.07 Purified water q.s. for 100 2. 10%aqueous solution peptide extract of lupin + fluorides Peptide extract oflupin 2 Sodium monofluorophosphate 0.75 Sodium fluoride 0.10 70%sorbitol 35 Synthetic silica with strong 13 abrasive power Syntheticsilica with weak 5 abrasive power Sodium carboxymethylcellulose 1.6Sodium lauryl sulfate 1 Mentholated flavoring 0.85 Titanium oxide 0.5Sodium hydroxide lye 0.5 Sodium cyclamate 0.3 Menthol 0.15 Sodiumsaccharin 0.07 Purified water q.s. for 100

What is claimed is:
 1. A method of treating or preventing a condition ordisease linked to degradation of collagen or another extracellularsupport macroprotein, or a disease linked to excessive expression of aproteolytic enzyme in a mammal, comprising administering a compositioncomprising a hydrolyzed peptide extract of lupin to a mammal.
 2. Themethod of claim 1 wherein the composition is administered orally.
 3. Themethod of claim 1 wherein the composition is administered topically. 4.The method of claim 1 wherein the condition or disease linked toexcessive or pathological degradation of collagen or anotherextracellular support macroprotein, or a disease linked to excessiveexpression of a proteolytic enzyme is neoangiogenesis, psoriasis, atumor, erythosis, acne erythemotosa, rosacea, arthrosis, parodontosis, aperiodontal disease, a skin lesion, an inflammatory disease, acicatrization deficiency, an ulcer, a bum, a blistering disease, or anattack of dental enamel.
 5. The method of claim 4 wherein the conditionor disease is a skin lesion due to aging, or a skin lesion caused bysolar radiation, tobacco use, pollution or stress.
 6. The method ofclaim 1 wherein the composition inhibits metalloprotease activity whenadministered to the subject.
 7. The method of claim 1 wherein thecomposition inhibits collagenase activity, gelatinase activity orstromelysin activity when administered to the subject.
 8. The method ofclaim 1 wherein the hydrolyzed peptide extract of lupin is a hydrolyzedextract of a sweet white lupin.
 9. The method of claim 8 wherein thehydrolyzed peptide extract of the sweet white lupin is a hydrolyzedextract of an Ares variety of sweet white lupin.
 10. The method of claim1 wherein the mammal is a human.
 11. A method of inhibitingmetalloprotease activity in a mammal, comprising administering ahydrolyzed peptide extract of lupin to the mammal.
 12. The method ofclaim 11, wherein the metalloprotease activity is collagenase activity,gelatinase activity or stromelysin activity.
 13. The method of claim 111wherein the hydrolyzed peptide extract of lupin is a hydrolyzed extractof a sweet white lupin or a hydrolyzed extract of an Ares variety ofsweet white lupin.
 14. The method of claim 11 wherein the mammal is ahuman.